Mitochondrial dysfunction and oxidative stress are consistent features of Alzheimer's disease (AD). The reactive oxygen species-mediated damage and mitochondrial dysfunction may be causally related. The mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) is consistently deficiently in AD, and appears to be particularly vulnerabl4e to oxidative stress-related insults. Genetic studies since the previous submission appear to have confirmed, in four different studies with a total of over 1400 subjects, an association between AD and a polymorphism of the DLST gene that encodes the E2K subunit of KGDHC. The genetic disequilibrium has been shown in members from 40 Swedish AD families, and in "sporadic" Caucasian (Appendix 1) and Japanese subjects. These findings have led to the following hypotheses: 1) The deficiency of KGDHC in AD is usually due to the susceptibility of KGDHC to oxidative stress. 2) A genetic anomaly of DLST may be a risk factor for AD in a subgroup of patients. Biochemical studies (Aim 1) will identify the vulnerable subunit, and the crucial amino acids or prosthetic groups responsible for the vulnerability of KGDHC in AD. The approach requires quantitation of each of the three KGDHC and recombinant human KGDHC subunits. We will measure the enzyme activities and protein immunochemical levels of the three KGDHC subunits to identify the subunit vulnerability, and use mass spectroscopy and amino aid sequencing to help identify the crucial amino acids or prosthetic groups. Genetic search (Aim 2) for pathogenic mutations in DLST will test primarily familial cases from the Swedish AD families, and also selected "sporadic" cases from the Caucasian Jewish cohort who possess the 'at risk" DLST genotype. A candidate, if found in familial AD cases, will be tested for genetic linkage in affected Swedish families. The biochemical tests (Aim 1) will also be used in Project 3 to define the responses of KGDHC proteins to various metabolic insults and under KGDHC deficiency conditions in cultured cell stems. If a plausible DLST mutation is found (Aim 2), then the consequence of that mutation will be tested using recombinant mutant E2k and using test parameters developed in Aim 1.